A major problem in the cancer chemotherapy is the development of resistance to a whole range of drugs not only similar to the drugs used for resistance induction but also to some functionally and structurally unrelated. It's one of the multifactorial causes of failure of chemotherapy. Thus it appears essential to evaluate the multi-drug resistance (MDR) in living cells populations to: detect the MDR phenotype, to discriminate between resistant and sensitive cells, to identify mechanisms which are involved in the induction or the reversion of resistance and to study the cytotoxic process. Such a challenge implies the use of multiparametric approach that has been possible using a protocol involving microfluorometry connected to numerical image analysis on single living cells. This protocol relays on the correlation existing between the decreased intracellular accumulation of some fluorescent probes such as Hoechst 33342 (Ho342) and Rhodamine 123 (R123) in resistant cells. The simultaneous estimation of the fluorescence intensities of these probes has required the use of a third probe, the Nile Red, for cell contour delineation. The analysis of parameters related to Ho342 and R123 allows the discrimination of sensitive and resistant cells. So the multiparametric approach using multi-wavelength image analysis, which appears to be a powerful technique, has allowed us to show on human lymphoblastoid CCRF-CEM cells lines that the cytotoxic effects could be different depending on the cell resistance or on the cytotoxic drug used: Adriamycine, Vinblastine and the different cell behavior could be used for cell differentiation.